The Na+/Cl-dependent and cocaine-sensitive neurotransmitter transporters include the dopamine transporter the norepinephrine transporter and the serotonin transporter. These transporters play a critical role in regulating the amount of available neurotransmitter in the synaptic cleft by mediating rapid reuptake of the neurotransmitter to the presynaptic nerve terminal. Blocking this transport process represents the principle mechanism underlying the psychostimulatory effects and abuse potential of cocaine and related drugs. The current knowledge about how this class of transporters works at the molecular level is remarkably small. Fundamental questions such as the structural basis for the inhibitory action of cocaine and the nature of the molecular processes responsible for the substrate translocation mechanism remain unanswered. It is the long term goal of the present project to define the structural basis for the action of cocaine and related psycho-stimulants at Na+/Cl dependent neurotransmitter transporters. We will seeks to accomplish this goal by obtaining insight into the secondary and tertiary structure of Na+/Cl-dependent neurotransmitter transporters by characterizing the biophysical nature of the cocaine binding site and by identifying distinct functional states by characterizing the biophysical nature of the cocaine binding site and by identifying distinct functional states and delineating the conformational changes responsible for the transport process. In our experimental strategy we will take advantage of unique methodological approaches, such as Zn2+-site engineering and fluorescence spectroscopy techniques. Our recent identification of two histidines forming two coordinates in an endogenous Zn2 binding site in the dopamine transporter has defined the first intramolecular distance constraint in the tertiary structure of a Na+/Cl-dependent transporter. These coordinates will serve as a critical scaffold for systematic design of new artificial Zn2+ sites that ultimately, and in conjunction with the other components of the present PPG, should lead to a low-resolution model of the secondary and tertiary structure of Na+/Cl-dependent fluorescent probes, site-selectively incorporated into the transporter molecule, as molecular reporters of conformational changes involved in substrate translocation and cocaine action. In addition, we will use a series of fluorescent cocaine analogues as a tool to characterize the biophysical characteristics of the cocaine binding site and its relatedness permeation pathway. Importantly, the challenge of our goals defines the demand for a methodologically integrated effort between the different components of the present PPG.